Scaffolding Proteins in Myofibril Assembly: N-RAP is a striated muscle-specific scaffolding protein found in developing myofibrils, and is thought to organize actin and alpha-actinin into premyofibril complexes. In developing skeletal and cardiac muscle and cultured myotubes and cardiomyocytes, N-RAP protein is primarily associated with developing myofibrillar structures containing &#945;-actinin, but is not present in mature myofibrils. Using primary cardiomyocyte cultures as a model system, we have demonstrated that N-RAP assembles into an actin containing scaffold;that alpha-actinin is recruited to the complex following N-RAP incorporation;that periodicity is established in the premyofibril before alpha-actinin recruitment;that the N-RAP scaffold is much more stable than the assembling structural components;that N-RAP dynamics increase as assembly progresses;and that N-RAP leaves the structure after assembly is complete. N-RAP also binds to Krp1, a kelch-repeat protein hypothesized to promote myofibril assembly through interaction with N-RAP and actin. Our previous work demonstrated that myofibril maturation was interrupted by Krp1 knockdown in cultured cardiomyocytes, leading to the accumulation of very thin myofibrils and their subsequent degradation. We concluded that Krp1 promotes lateral fusion of adjacent thin fibrils into mature, wide myofibrils but is not necessary for longitudinal organization of actin and myosin filaments. In order to discover the molecular mechanism by which Krp1 promotes lateral fusion of myofibril assembly intermediates, we are studying the targeting and binding properties of individual regions of Krp1. To that end we have subcloned the Krp1 kelch repeats, the Krp1 BTB domain, and the Krp1 BACK domain into vectors for expression as YFP-fusion proteins and GST fusion proteins for cell biological and biochemical studies. When YFP-tagged domains of Krp1 were expressed in primary cardiomyocytes, no obvious localization of the constructs was observed. When the GST-Krp1 fusion protein was incubated with lysate derived form cultured muscle cells and the bound proteins analyzed by gel electrophoresis, several prominant protein bands were observed. We are currently identfying these potential Krp1 binding partners by mass spectroscopy. Creation and Characterization of N-RAP Overexpressing Transgenic Mice: Our previous collaborative work has shown that N-RAP expression increases in genetic mouse models of dilated cardiomyopathy, and that the increased N-RAP expression in these animals occurs shortly after birth and precedes other molecular changes in the heart. We are directly testing the effects of N-RAP overexpression using transgenic mouse lines that overexpress N-RAP in heart using the well-characterized tet-off system. We cloned full length N-RAP into the pTRE2hyg2-myc tet-responsive vector, excised the transgene, and used this DNA construct to produce transgenic founder mice. The resulting tet-responsive N-RAP transgenic mice were mated with cardiac-specific tTA expressing mice. The double transgenic progeny were screened for myc-N-RAP expression. We have identified three founder lines that exhibit tTA-dependent cardiac-specific expression of the N-RAP transgene at both the mRNA and protein level. Myc-N-RAP transgene expression gives rise to elevated total (endogenous + transgenic) N-RAP protein detected with anti-N-RAP antibody. The data show that these lines exhibit total cardiac N-RAP levels ranging from 1.2 to 2.6 times that of wild type animals. The extent of N-RAP overexpression observed in two of these lines is similar to that observed in adult MLP knockout mice, where dilated cardiomyopathy is associated with N-RAP levels 2.5 times normal. Immunofluorescence shows that myc-N-RAP, the protein product of the transgene, is concetrated at the cardiac intercalated disk. Focusing on the two lines exhibiting the most N-RAP overexpression, we are characterizing the hearts of 8-12 week old double transgenic mice in vivo using echocardiogrpahy and magnetic resonance imaging and in vitro by standard histological methods. In 8-12 week old mice, N-RAP overexpression had no effect on right and left ventricular wall thicknesses, fractional shortening and ejection fractions. We have begun characterizing the hearts of 10 month old transgenic mice in order to determine if prolonged overexpression of N-RAP affects cardiac structure and function. Myofibril Function in GAA Knockout Mice: Pompe disease, a deficiency of lysosomal acid &#945;-glucosidase (GAA), is a disorder of glycogen metabolism that affects infants, juveniles or adults. In all forms of the disease there is progressive muscle pathology leading to early death. The pathology - accumulation of glycogen in lysosomes and autophagic buildup - is well characterized, but how it leads to profound muscle weakness is unknown. The purpose of the present investigation was to characterize the mechanical and biochemical properties of skeletal muscle from the GAA knockout mice, a model of human Pompe disease. The study was performed on isolated myofibers derived from severely affected fast glycolytic muscle, which had both major pathologic features. Psoas muscle fibers were first demembranated so that the contractile proteins could be directly relaxed or activated by controlling the composition of the bathing solution. The results revealed that severe atrophy found in the muscle was not the only factor that contributed to muscle weakness. Tension generated by single fibers activated with calcium, when normalized by the fiber cross-sectional area, was decreased 46 % in the KO mice compared with the wild type. On the other hand, concentrations of the contractile proteins myosin and actin remained unchanged, while the total protein concentration was slightly higher in the KO mice. Structurally, x-ray diffraction showed that the actin and myosin filaments within the cytoplasm, normally arranged in a hexagonal array, were highly disordered in the GAA knockout muscle, and a lower fraction of myosin cross-bridges was detected near the actin filaments in the relaxed muscle. The results are consistent with a disruption of actin and myosin interactions in the KO muscle, resulting in reduced tension output from the myofibrils.